This is an R21 exploratory/development grant application to develop methods to quantify the content of alcohol metabolizing enzymes and variants (initially alcohol dehydrogenase and eventually aldehyde dehydrogenase and microsomal ethanol oxidizing CYP isoenzymes) in human tissues (liver and esophagus). We believe that the application fits the R21 guidelines because we will use newly developed mass spectrometry proteomics methods to address the specific aims. The application is also in response to PA-05-074, Mechanisms of Alcohol-Induced Tissue Injury, because it provides important protein expression information to support the investigation of alcohol metabolizing enzyme genotypes that confer susceptibility or resistance to ethanol-induced cell/tissue damage. Our central hypothesis is that the expression/content of alcohol metabolizing enzymes and variants determine the pharmacokinetics and cellular toxicology of ethanol and acetaldehyde in tissues. In Aim #1, we will develop proteomics methods to quantify the expression of seven alcohol dehydrogenase (ADH) isoenzymes in tissues by triple quadrupole linear ion trap [unreadable] LC/MS/MS and multiple reactions monitoring analysis with appropriate peptide/protein internal standards. We will assess the feasibility of developing methods to look at the whole set of ADH, ALDH and microsomal ethanol oxidizing CYP isoenzymes involved in alcohol metabolism simultaneously. In Aim #2, we will collect and genotype human liver samples and matched normal esophagus and squamous cell carcinoma tissue samples from a local population in Indianapolis for the pilot proteomics study. The tissue samples will be genotyped at ADH and other alcohol metabolizing enzyme loci. In Aim #3, we will perform a preliminary proteomics study to look at the variability of expression/content of the seven ADH isoenzymes in the liver and esophagus tissue samples using the proteomics methods developed in Aim #1. We will identify whether there is a correlation between ADH polymorphisms and protein expression in tissues. We will estimate the individual contribution of the seven ADHs to alcohol metabolic capacity in tissue samples and the contribution of polymorphism to differences in alcohol metabolic capacity. We will assess the feasibility of performing targeted proteomics studies with tissues from other populations that express different SNPs in alcohol metabolizing enzymes. [unreadable] [unreadable] [unreadable]